System and methods for providing corrected analyte concentration measurements

ABSTRACT

Methods and devices for determining the concentration of a constituent in a physiological sample are provided. The physiological sample is introduced into an electrochemical cell having a working and counter electrode. At least one electrochemical signal is measured based on a reaction taking place at the cell. The preliminary concentration of the constituent is then calculated from the electrochemical signal. This preliminary concentration is then multiplied by a hematocrit correction factor to obtain the constituent concentration in the sample, where the hematocrit correction factor is a function of the at least one electrochemical signal. The subject methods and devices are suited for use in the determination of a wide variety of analytes in a wide variety of samples, and are particularly suited for the determination of analytes in whole blood or derivatives thereof, where an analyte of particular interest is glucose.

FIELD OF THE INVENTION

The present invention relates to the field of diagnostic testing and,more particularly, to diagnostic testing systems for measuring theconcentration of a substance in a sample.

BACKGROUND OF THE INVENTION

The present disclosure relates to a biosensor system for measuring ananalyte in a bodily fluid, such as blood, wherein the system comprises aunique process and system for correcting inaccuracies in sampleconcentration measurements. For example, the present disclosure providesmethods of correcting analyte concentration measurements of bodilyfluids.

Electrochemical sensors have long been used to detect and/or measure thepresence of substances in a fluid sample. In the most basic sense,electrochemical sensors comprise a reagent mixture containing at leastan electron transfer agent (also referred to as an “electron mediator”)and an analyte specific bio-catalytic protein (e.g. a particularenzyme), and one or more electrodes. Such sensors rely on electrontransfer between the electron mediator and the electrode surfaces andfunction by measuring electrochemical redox reactions. When used in anelectrochemical biosensor system or device, the electron transferreactions are transformed into an electrical signal that correlates tothe concentration of the analyte being measured in the fluid sample.

The use of such electrochemical sensors to detect analytes in bodilyfluids, such as blood or blood derived products, tears, urine, andsaliva, has become important, and in some cases, vital to maintain thehealth of certain individuals. In the health care field, people such asdiabetics, for example, have a need to monitor a particular constituentwithin their bodily fluids. A number of systems are available that allowpeople to test a body fluid, such as, blood, urine, or saliva, toconveniently monitor the level of a particular fluid constituent, suchas, for example, cholesterol, proteins, and glucose. Patients sufferingfrom diabetes, a disorder of the pancreas where insufficient insulinproduction prevents the proper digestion of sugar, have a need tocarefully monitor their blood glucose levels on a daily basis. A numberof systems that allow people to conveniently monitor their blood glucoselevels are available. Such systems typically include a test strip wherethe user applies a blood sample and a meter that “reads” the test stripto determine the glucose level in the blood sample. For example, testingand controlling blood glucose for people with diabetes can reduce theirrisk of serious damage to the eyes, nerves, and kidneys.

An exemplary electrochemical biosensor is described in U.S. Pat. No.6,743,635 ('635 patent) which is incorporated by reference herein in itsentirety. The '635 patent describes an electrochemical biosensor used tomeasure glucose level in a blood sample. The electrochemical biosensorsystem is comprised of a test strip and a meter. The test strip includesa sample chamber, a working electrode, a counter electrode, andfill-detect electrodes. A reagent layer is disposed in the samplechamber. The reagent layer contains an enzyme specific for glucose, suchas, glucose oxidase, and a mediator, such as, potassium ferricyanide orruthenium hexaamine. When a user applies a blood sample to the samplechamber on the test strip, the reagents react with the glucose in theblood sample and the meter applies a voltage to the electrodes to causeredox reactions. The meter measures the resulting current that flowsbetween the working and counter electrodes and calculates the glucoselevel based on the current measurements.

In biosensors that measure a particular constituent level in blood,certain components of the blood can undesirably affect the measurementsand lead to inaccuracies in the detected signal. This inaccuracy mayresult in an incorrect reading, leaving the patient unaware of apotentially dangerous blood sugar level, for example. As one example,the particular blood hematocrit level (i.e. the percentage of the amountof blood that is occupied by red blood cells) can erroneously affect aresulting analyte concentration measurement.

It is known that variations in the volume of red blood cells can causeerrors in the glucose readings measured with disposable electrochemicaltest strips. Typically, a negative bias (i.e., lower calculated analyteconcentration) is observed at high hematocrits, while a positive bias(i.e., higher calculated analyte concentration) is observed at lowhematocrits (a condition representative of an anemic state). At highhematocrits, for example, the red blood cells may (1) impede thereaction of enzymes and electrochemical mediators, (2) reduce the rateof chemistry dissolution since there less plasma volume to solvate thechemical reactants, and (3) slow down diffusion of the mediator. Thesefactors can result in a lower than expected glucose reading as lesscurrent is produced during the electrochemical process. Conversely, atlow hematocrits there are less red blood cells interfering with theelectrochemical reaction than expected and a higher current can bemeasured. Since the concentration of red blood cells alters thediffusion of dissolved reactants, faradaic current measurements areimpacted. In addition, the blood sample resistance is also hematocritdependent, which can affect charging current measurements.

Several strategies have been used to reduce or avoid hematocrit basedvariations on blood glucose readings. For example, test strips have beendesigned incorporating meshes to remove red blood cells from the samplesor have included particles in chemistry formulations in order toincrease the viscosity of red blood cell and remove the effect of lowhematocrits. These methods have the disadvantages of increasing the costand complexity of test strips and undesirably increase the time requiredfor accurate glucose measurement. In addition, alternating current (AC)impedance methods have also been developed to measure electrochemicalsignals at frequencies independent of and hematocrit effect. Suchmethods suffer from the increased cost and complexity of advanced metersrequired for signal filtering and analysis.

An additional prior hematocrit correction scheme is described in U.S.Pat. No. 6,475,372. In that method, a two potential pulse sequence isemployed to estimate an initial glucose concentration and determine amultiplicative hematocrit correction factor. A hematocrit correctionfactor is a particular numerical value or equation that is used (suchas, for example, by taking the product of the initial measurement andthe determined hematocrit correction factor) to correct an initialconcentration measurement. More specifically, a first pulse of onepolarity is applied to the reaction cell with the sample, followed by asecond pulse of an opposite polarity to the reaction cell with thesample.

The current responses resulting from both pulses are measured as afunction of time, with pulse widths for the first step ranging fromabout 3 to 20 seconds, and for the second step from 1 to 10 s. Theglucose concentration in the sample is then estimated from the measuredcurrent-time transients (i.e. the current response). A blood hematocritcorrection factor is determined using statistical methods, such as, fromthe mathematical fit of a three dimensional plot based on data collectedat several glucose concentrations and blood hematocrit levels.

The three dimensional plot is created from the following variables: theratio of the first average current response value to the second averagecurrent response value, the estimated glucose concentration, and theratio of the YSI determined glucose concentration to the estimatedglucose concentration minus a background value. The initial estimatedglucose concentration is then multiplied by the calculated bloodhematocrit correction factor to determine the reported glucoseconcentration.

Using the process of U.S. Pat. No. 6,475,372, most data points werefound to fall within +/−15% of actual glucose concentrations using thehematocrit correction factor equation. Data processing using thistechnique, however, is still fairly complicated because both ahematocrit correction factor and an estimated glucose concentration mustbe determined to establish the corrected glucose value. In addition, thetime duration of the first step greatly increases the overall test timeof the biosensor, which is undesirable from the user's perspective.

Accordingly, novel systems and methods for providing corrected analyteconcentration measurements are desired that overcome the drawbacks ofcurrent biosensors and improve upon existing electrochemical biosensortechnologies so that measurements are more accurate.

SUMMARY OF THE INVENTION

Embodiments of the present invention are directed to medical devices forimmobilization and/or retrieval of objects within anatomical lumens ofthe body that obviate one or more of the limitations and disadvantagesof prior immobilization and retrieval devices.

One embodiment is directed to a biosensor including a sample receptionregion for receiving a blood sample and a reaction reagent system. Thereaction reagent system includes an oxidation-reduction enzyme specificfor the constituent; a first electron mediator capable of beingreversibly reduced and oxidized such that a first electrochemical signalresulting from the reduction or oxidation is related to the constituentconcentration in the blood sample; and a second electron mediatorcapable of undergoing an electrochemical redox reaction where a secondelectrochemical signal produced by oxidation or reduction of the secondmediator is not directly related to the constituent concentration in theblood sample. The second electrochemical signal changes based on thehematocrit level of the blood sample.

In various embodiments, the biosensor may include one or more of thefollowing additional features: wherein the constituent is glucose;wherein the first mediator is a ruthenium containing material; whereinthe ruthenium containing material comprises hexaamine ruthenium (III)trichloride; wherein the second mediator comprises brilliant cresylblue; wherein the second mediator comprises gentisic acid(2,5-dihydroxybenzoic acid); wherein the second mediator comprises2,3,4-trihydroxybenzoic acid; wherein the second mediator does notinterfere with the first electrochemical signal; wherein the secondmediator is oxidized or reduced in a potential range distinguishablefrom that of the first mediator; wherein the second electron mediator isoxidized or reduced at a potential having a magnitude at least 0.2 voltsgreater or less than that used to oxidize or reduce the first electronmediator; wherein the first and second electrochemical signals areelectric current signals obtained through multi-step chronoamperometry;wherein the first and second electrochemical signals are electriccurrent signals obtained through square wave voltammetry; wherein thefirst and second electrochemical signals are electric current signalsobtained through differential pulse amperometry; and wherein the firstand second electrochemical signals are electric current signals obtainedthrough cyclic voltammetry.

Another embodiment of the invention is directed to a method fordetermining a constituent concentration in blood including introducingthe blood sample into an electrochemical cell. The electrochemical cellmay comprise spaced apart working and counter electrodes and a redoxreagent system comprising an enzyme. The cell also includes a firstelectron mediator capable of being reversibly reduced and oxidized suchthat a first electrochemical signal resulting from the reduction oroxidation is related to the constituent concentration in the bloodsample. The cell also includes a second electron mediator capable ofcapable of undergoing an electrochemical redox reaction where a secondelectrochemical signal produced by oxidation or reduction of the secondmediator is not directly related to the constituent concentration in theblood sample and changes based on the hematocrit level of the bloodsample. The method further includes obtaining the first electrochemicalsignal; obtaining the second electrochemical signal; determining aninitial value corresponding to the constituent concentration of thesample using data from the first electrochemical signal; and correctingthe initial value corresponding to the constituent concentration of thesample to remove an effect of the hematocrit level of the sample using astatistical correlation algorithm and data from the secondelectrochemical signal.

In various embodiments, the method may include one or more of thefollowing additional features: wherein the constituent is glucose;wherein correcting the initial value comprises deriving a preliminaryconstituent concentration from the first and second signals andmultiplying the preliminary constituent concentration by a correctionfactor based on the second electrochemical signal to derive theconstituent concentration in the sample, corrected to offset an effectof the hematocrit level of the blood sample; wherein the statisticalcorrelation comprises determining a slope of the second electrochemicalsignal; wherein the statistical correlation comprises determining aslope of both the first and second electrochemical signals; wherein thefirst electrochemical signal is obtained by applying to theelectrochemical cell, a first electric potential of a magnitude capableof oxidizing or reducing the first electron mediator and not capable ofoxidizing or reducing the second electron mediator; wherein the secondelectrochemical signal is obtained by applying to the electrochemicalcell, a second electric potential of a magnitude capable of oxidizing orreducing the second electron mediator and not capable of oxidizing orreducing the first electron mediator; wherein the second electronmediator is oxidized or reduced at a potential having a magnitude atleast 0.2 volts greater or less than that used to oxidize or reduce thefirst electron mediator; wherein obtaining the first and secondelectrochemical signals comprises using multi-step chronoamperometry;wherein obtaining the first and second electrochemical signals comprisesusing square wave voltammetry; wherein obtaining the first and secondelectrochemical signals comprises using differential pulse amperometry;wherein obtaining the first and second electrochemical signals comprisesusing cyclic voltammetry; wherein the second electron mediator comprisesbrilliant cresyl blue; wherein the second electron mediator comprisesgentisic acid (2,5-dihydroxybenzoic acid); and wherein the secondelectron mediator comprises 2,3,4-trihydroxybenzoic acid.

Another embodiment of the invention is directed to a method fordetermining the hematocrit corrected concentration of an analyte in aphysiological sample comprising introducing the physiological sampleinto an electrochemical cell. The electrochemical cell may comprisespaced apart working and counter electrodes and a redox reagent systemcomprising an enzyme and a mediator. The method also includes applying afirst electric potential to the reaction cell and measuring cell currentduring a first 50 milliseconds of the first electric potential as afunction of time to obtain a first time-current transient; applying asecond electric potential to said cell, and measuring cell current as afunction of time to obtain a second time-current transient; deriving apreliminary analyte concentration from said first and secondtime-current transients; and multiplying the preliminary analyteconcentration by a hematocrit correction factor based on the first andsecond time-current transient to derive the hematocrit corrected analyteconcentration in said sample whereby the hematocrit correctedconcentration of said analyte in said sample is determined.

In various embodiments, the method may include one or more of thefollowing additional features: wherein the first electric potential is anegative electric pulse and the second electrical potential is apositive electrical pulse; wherein the first electric potential is anapplied pulse having a duration of about 1-10 milliseconds; wherein thepreliminary analyte concentration is determined in part based on acurrent time transient value as sampled at an end of the applied pulseof the first electric potential; wherein the second electric potentialis an applied pulse or about 1-4 seconds; and wherein the preliminaryanalyte concentration is determined in part based on a current timetransient value as sampled at an end of the applied pulse of the secondelectric potential.

Another embodiment of the invention is directed to a method formanufacturing a plurality of test strips, comprising forming a webcontaining a conductive layer and a base layer and partially forming theplurality of test strips by electrically isolating a first group ofconductive components in the conductive layer using a first process. Themethod further includes subsequently forming the plurality of teststrips by electrically isolating a second group of conductive componentsin the conductive layer using a second process wherein first and secondprocesses are not the same. The method also includes forming a reagentlayer including an enzyme; a first electron mediator capable of beingreversibly reduced and oxidized such that a first electrochemical signalresulting from the reduction or oxidation is related to the constituentconcentration in the blood sample; and a second electron mediatorcapable of undergoing an electrochemical redox reaction where a secondelectrochemical signal produced by oxidation or reduction of the secondmediator is not directly related to the constituent concentration in theblood sample and changes based on the hematocrit level of the bloodsample.

In various embodiments, the method may include one or more of thefollowing additional features: wherein the web includes a plurality ofregistration points; wherein the first process includes a laser ablationprocess; wherein the second process includes a separation process;wherein the separation process includes stamping; wherein the separationprocess includes separating a plurality of test strips from the web;wherein the plurality of registration points are separated byapproximately 9 mm; wherein the plurality of registration points areseparated by less than approximately 9 mm; and wherein the first groupof conductive components are separated by less than approximately 9 mm.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cyclic voltammogram associated with the use of a goldelectrode with a ruthenium hexaamine electron mediator.

FIG. 2 is a graph depicting the change in current response over timeduring the application of an input voltage pulse in a samplemeasurement.

FIG. 3A provides a quadratic fit surface plot from measured data and YSImeasured concentration values derived from samples at multiple analyteconcentrations and blood hematocrit levels.

FIG. 3B provides a graph depicting the percent bias of calculatedglucose concentration values compared with YSI measured sampleconcentration values at various hematocrit levels and analyteconcentration levels.

FIG. 3C provides a best fit surface plot from measured data and %deviation values from YSI glucose concentration values derived fromsamples at multiple analyte concentrations and blood hematocrit levels.

FIG. 3D is a graph depicting the percent bias of corrected glucosevalues from YSI measured sample concentration values at varioushematocrit levels and analyte concentration levels, according to oneembodiment of the invention.

FIG. 4A provides a Taylor series fit surface plot from measured data andYSI measured concentration values derived from samples at multipleanalyte concentrations and blood hematocrit levels in a single pulsemethod.

FIG. 4B is a graph depicting the percent bias of calculated glucoseconcentration values from YSI measured sample concentration values atvarious hematocrit levels and analyte concentration levels in a singlepulse method.

FIG. 5 is a graph depicting the relationship between a particularamperometricly derived ratio and the particular blood sample hematocritlevel at various analyte concentration levels.

FIG. 6 is a graph depicting the relationship between a particularamperometricly derived ratio and the YSI measured sample concentrationvalues.

FIG. 7 is a cyclic voltammogram associated with an SRP electronmediator, according to an embodiment of the present disclosure.

FIG. 8A is a linear sweep voltammogram associated with another SRPelectron mediator, according to an embodiment of the present disclosure.

FIG. 8B depicts two linear sweep voltammograms, comparing the SRPelectron mediator of FIG. 8A with another SRP electron mediator,according to an embodiment of the present disclosure.

FIG. 8C is a table depicting corrected measurement values using oneparticular SRP substance.

FIG. 9 depicts a particular potential input waveform applied at theworking electrode relative to a counter electrode, according to anembodiment of the present disclosure.

FIG. 10 is a graph depicting the change in current response over timeduring the application of the input waveform of FIG. 9 in a samplemeasurement using a primary redox probe and a secondary redox probe(“SRP”).

FIG. 11 is another graph depicting the change in current response overtime during the application of the waveform described in FIG. 9.

FIG. 12 is a table depicting corrected measurement values using a firstcorrection algorithm.

FIG. 13 is a table depicting corrected measurement values using a secondcorrection algorithm.

FIG. 14 is a graph depicting the dependence of an SRP mediator on theparticular hematocrit level of blood.

FIG. 15 depicts the relationship between the measured analyte signalmagnitude and the actual sample analyte concentration at multipleconcentrations of the SRP.

FIG. 16 is a graph showing the derived relationship between a calculatedSRP factor and the hematocrit of the sample.

FIG. 17 is a top plan view of a test strip according to an illustrativeembodiment of the invention.

FIG. 18 is a cross-sectional view of the test strip of FIG. 17, takenalong line 2-2.

FIG. 19 is a top view of a reel or web according to a furtherillustrative embodiment of the invention.

FIG. 20 is a top view of a card formed from a portion of the reel or webaccording to a further illustrative embodiment of the invention.

FIG. 21 is a top view of a conductive layer according to an illustrativeembodiment of the invention.

FIG. 22 is a top view of a dielectric layer according to an illustrativeembodiment of the invention.

FIG. 23 is a diagram of the manufacturing process according to a furtherillustrative embodiment of the invention.

DESCRIPTION OF THE EMBODIMENTS

In accordance with the present disclosure provided herein areelectrochemical biosensors developed for measuring an analyte in anon-homogenous fluid sample, such as in a food product or in a bodilyfluid chosen from blood, urine, saliva and tears. At a minimum, thebiosensor includes at least one or more electrodes and a reactionreagent system comprising an electron mediator and anoxidation-reduction enzyme specific for the analyte to be measured. Inone embodiment, the electron mediator comprises a ruthenium containingmaterial, such as ruthenium hexaamine (III) trichloride.

As used herein, the phrase “working electrode” is an electrode at whichthe electrochemical oxidation and/or reduction reaction occurs, e.g.,where the analyte, typically the electron mediator, is oxidized orreduced.

“Counter electrode” is an electrode paired with the working electrode. Acurrent of equal magnitude and of opposite polarity to the workingelectrode passes through the counter electrode.

“YSI” or “YSI values” means a particular analyte concentration asdetermined using a Yellow Springs Instrument glucose analyzer, such as,for example, the YSI model 2300 Stat Plus.

As noted above, the '635 patent describes an exemplary electrochemicalbiosensor used to measure glucose level in a blood sample. Theelectrochemical biosensor system is comprised of a test strip and ameter. The test strip includes a sample chamber, a working electrode, acounter electrode, and fill-detect electrodes. A reagent layer isdisposed in the sample chamber, and generally covers at least part ofthe working electrode as well as the counter electrode. The reagentlayer contains an enzyme specific for glucose, such as, glucose oxidaseor glucose dehydrogenase, and a mediator, such as, potassiumferricyanide or ruthenium hexamine.

In one example, glucose oxidase is used in the reagent layer. Therecitation of glucose oxidase is intended as an example only and othermaterials can be used without departing from the scope of the invention.For example, glucose dehydrogenase is another enzyme that is used inglucose biosensors. Similarly, while potassium ferricyanide is listed asa possible mediator, other possible mediators are contemplated. Forexample, additional mediators include, but are not limited to,ruthenium, osmium, and organic redox compounds. In one embodiment,during a sample test, the glucose oxidase initiates a reaction thatoxidizes the glucose to gluconic acid and reduces the ferricyanide toferrocyanide. When an appropriate voltage is applied to a workingelectrode, relative to a counter electrode, the ferrocyanide is oxidizedto ferricyanide, thereby generating a current that is related to theglucose concentration in the blood sample. The meter then calculates theglucose level based on the measured current and displays the calculatedglucose level to the user.

Commonly owned co-pending U.S. patent application Ser. No. 11/242,925(which is incorporated herein by reference in its entirety) disclosesthe use of ruthenium hexaamine as another potential mediator. Whenruthenium hexaamine [Ru(NH₃)₆]³⁺ is used, the glucose oxidase initiatesa reaction that oxidizes the glucose to gluconic acid and reduces[Ru(NH₃)₆]³⁺ to [Ru(NH₃)₆]²⁺. In the case of glucose dehydrogenase, theenzyme oxidizes glucose to glucono-1,5-lactone, and reduces [Ru(NH₃)₆]³⁺to [Ru(NH₃)₆]²⁺. When an appropriate voltage is applied to the workingelectrode, relative to the counter electrode, the electron mediator isoxidized. For example, ruthenium hexaamine [Ru(NH₃)₆]²⁺ is oxidized to[Ru(NH₃)₆]³⁺, thereby generating a current that is related to theglucose concentration in the blood sample.

The systems and methods of the present application rely on electrontransfer between the electron mediator and the electrode surfaces andfunction by measuring electrochemical redox reactions. As noted above,these electron transfer reactions (such as the ferrocyanide or rutheniumhexaamine reactions described above) are transformed into an electricalsignal that correlates to the concentration of the analyte beingmeasured in the fluid sample. More particularly, the electrical signalresults from the application of particular electrode potential input(comprised of a single constant pulse or distinct separate pulses atmore than one potential) at the working electrode relative to a counterelectrode.

The pulse or pulses are applied to the cell at a particularpredetermined potential relative to the redox potential of theparticular strip mediator used. As is known in the art, the redoxpotential of a substance is a measure (in volts) of the substancesaffinity for electrons (i.e. the substances electronegativity) comparedwith hydrogen, which is set at zero. Substances capable of oxidizinghydrogen have positive redox potentials. Substances capable of reducinghydrogen have negative redox potentials. One way to determine theparticular redox potential of a substance is by cyclic voltammetry. FIG.1, for example is a cyclic voltammogram associated with the use of agold electrode with a ruthenium hexaamine electron mediator. As seen inFIG. 1, the ruthenium hexaamine substance exhibits a redox potential ofabout −0.2 volts vs Ag/AgCl reference electrode in pH 7.25 phosophatebuffer solution.

Accordingly, where the desired electron transfer reaction is a reductionof the mediator, for example, a voltage pulse well negative of the redoxpotential is applied. Conversely, where the desired electron transferreaction is an oxidation of the mediator, a voltage pulse well positiveof the redox potential is applied. The particular electrode potentialinput into the cell results in an electric signal in the form of acurrent-time transient. In other words, the final concentrationmeasurement is based on the particular current-time transient (alsoknown as the amperometric current response) obtained as a result ofapplying a particular voltage potential to the cell (i.e. between theworking and counter electrodes) and observing the change in current overtime between the working and counter electrodes. FIG. 2, for example, isa graph depicting the change in current response over time during theapplication of an input voltage pulse in a sample measurement.

The electrochemical method described above with regard to U.S. Pat. No.6,475,372 is inherently based on a correction for the contribution ofhematocrit on the faradaic current generated by the negative input pulse(reduction of the mediator) or the positive input pulse (oxidation ofthe mediator that was reduced as part of the enzyme-glucose reaction).Initially, the current is composed of contributions from both thecharging of the electrical layers of the cell and the diffusion limited(faradaic) current. By the time the current of the first pulse issampled at time T=100 ms, the charging current has decayed and only thefaradaic current remains. The faradaic current can be generallydescribed by the Cottrell equation, Equation No. 1:i(t)=(nFAD ^(1/2) C ₀)/(π^(1/2) t ^(1/2))where n is the number of transferred electrons, F is Faraday's constant,A is the electrode area, D is the diffusion coefficient, and C₀ is theinitial analyte concentration. Since the effective diffusion coefficientof the analyte is dependent on hematocrit, the measured faradaic currentresponses of pulses 1 and 2 are used in the method of U.S. Pat. No.6,475,372 to model hematocrit dependence.

Faradaic vs. Charging Current Hematocrit Correction

The following aspect of the present invention provides anelectrochemical method to measure glucose with reduced hematocriteffect. In one embodiment of this method, a negative potential with apulse width of a few milliseconds (such as, for example, 1-10 ms, butincluding pulses of duration up to approximately 40 ms) is applied tothe electrochemical cell, followed by a positive potential having aduration of about 4 seconds (but including pulses of duration up to 10seconds). With regard to exemplary potential magnitudes, for rutheniumhexamine, a negative pulse ranging from approximately −0.2 to −0.45 Vmay be employed with a preferred potential of approximately −0.3 or−0.35 V. A second positive pulse may range from 0.2 to 0.4 V with apreferred potential of approximately 0.3 V. Naturally, the optimal rangeis directly related to the mediator. For example, if alternate mediatorsare utilized the optimal positive and negative potential pulses will berelated to the oxidation and reduction properties of this mediator.

The current is sampled near the end of both pulse widths. At the end ofthe first pulse, the charging current is a significant component of themeasured current. In general, electrochemical pulse methods have shownthat charging current exponentially decays to zero for most systemswithin 40 ms, while the faradaic current decays much more slowly. Thecharging current can be described by the following equation, EquationNo. 2:i=E/R _(s) *e[−t/(R _(s) *C _(d))]

where E is the applied potential, R_(s) is the solution resistance, andC_(d) is the electrode layer capacitance. Using this equation, both thesolution resistance variable and perhaps the capacitance is dependent onhematocrit and can therefore be manipulated via statistical analysis todetermine a hematocrit correction factor.

Accordingly, in the current method, the first pulse results in a currentresponse primarily described by Equation No. 2, while the second pulseresults in a current response primarily described by Equation No. 1,described above. By analyzing the first current response based onEquation No. 2, the hematocrit dependent variables of solutionresistance and capacitance can be analyzed via statistical analysis(e.g. with a best fit correlation, such as a Taylor Series fit) to helpdetermine a hematocrit correction factor. In addition, using the secondcurrent response and Equation No. 1, the hematocrit dependent variableof diffusion can also be analyzed via statistical analysis to assist indetermining a hematocrit correction factor.

Accordingly, in one embodiment of the current invention, a pulse 1current response, P1, (recorded, for example, at t=5 ms) and a pulse 2current response, P2, (recorded, for example, at t=4 s) are measuredresulting from tests performed on multiple fluid samples. These initialmeasurements may be performed using a particular lot of test strips.Then multiple samples, having known glucose concentration levels, aretested to determine and record the P1 and P2 current values for multipleglucose concentration values. These known glucose concentration levelsof the samples are then correlated with particular variables based onthe P1 and P2 data.

For example, FIG. 3A depicts a three-dimensional quadratic surface plotfit based on a correlation of P1 and P2 data collected at severalglucose concentrations and blood hematocrit levels. In FIG. 3A, thevariable of the P1 and P2 current ratio (P1/P2 as depicted along the Xaxis) and the variable of the P1 current (P1 as depicted along the Yaxis) are correlated (with a quadratic least squares best fit, forexample) with the known sample glucose concentration levels (the (mg/dl)concentration values along the Z axis) resulting in the surface plotdisplayed. Thereafter, for all strips in the given lot, the calculatingmeter is programmed with the corresponding surface plot fit. When usedto measure P1 charging current and P2 faradaic current of a particularsample, the meter calculates the appropriate glucose level according tothe surface plot correlation, which is then displayed on the meter.Alternate mathematical interpretations may be employed. For example, therelationship between P1 currents, P2 currents, and YSI or between P1/P2,P1, YSI may be correlated.

In a test experiment, test glucose concentration values for particularfluid samples were determined by inputting P1 and P1/P2 values into thequadratic fit equation. The resulting test data was compared with theactual YSI measured glucose concentration data for the fluid samples. Asseen in FIG. 3B, the samples tested included fluids having varyingglucose concentrations and varying hematocrit levels. The test valuesobtained were within +/−20% of the YSI measured glucose values, asdepicted in FIG. 3B.

Turning to FIG. 3C, one system of correcting for the hematocrit effecton analyte measurements is provided. The measured current is dependenton the % hematocrit in a given blood sample with higher currentsobserved for low hematocrits, and lower current observed at highhematocrits. Variations between the analyte concentrations calculated inthe procedure above and the actual YSI glucose values can bemathematically related to the P1 (5 ms)/P2 (4 s) values and the P1 (5ms) values in a three-dimensional plot. Other mathematicalconfigurations that relate the charging and faradaic currents to YSIdeviations may prove to be preferable to using alternate sensor designs(e.g. employing the ratio of pulse 1 and pulse 2, as described withreference to FIG. 5 below).

In FIG. 3C, the variable of the P1 current (P1 as depicted along the Xaxis) and the variable of the P1 and P2 current ratio (P1/P2 as depictedalong the Y axis) are correlated (with a quadratic least squares bestfit, for example) with the known value of the % deviation between theinitially calculated glucose concentration value and the YSI measuredglucose concentration levels (along the Z axis) resulting in the surfaceplot displayed. Using this surface plot, a particular % deviation isdetermined for each sample. These best fit surface plot correlations canthen be used to correct the glucose measured concentration to reduce theoffsetting effect of the particular blood hematocrit level.

In the approach using the plot of FIG. 3C, an estimated glucoseconcentration would be determined from the faradaic current measured atP2 (4 s). Next, the predicted percent deviation from YSI values would becalculated from the P1 (5 ms) current value to assess the extent ofhematocrit dependent effects. This value is then used to correct theestimated glucose concentration. The resulting corrected values aredepicted in FIG. 3D. More particularly, FIG. 3D provides the results ofa comparison between the corrected glucose concentration data and YSImeasured glucose concentration values. As seen in FIG. 3D, the bias ofthe corrected glucose values are depicted for samples at multipleconcentration values each at various hematocrit levels. Samples havingglucose concentration levels of 75 mg/dl, 150 mg/dl, 245 mg/dl, and 400mg/dl were tested, each at three different hematocrit percentage levels.The resulting % bias of calculated corrected glucose values deviatingfrom the YSI values are shown in FIG. 3D to be within +15% and −15%.

Table one, directly below, presents raw data from the above describedtwo-pulse methods of determining an analyte concentration using acharging vs. faradaic current measurement technique. TABLE 1 Two PulseData P1 (5 ms) P2 (4 s) Glucose HCT Ave StDev % CV Ave StDev % CV 75 201.16E−04 1.29E−05 11.14 2.91E−06 1.19E−07 4.10 75 40 1.03E−04 1.06E−0510.24 2.63E−06 9.08E−08 3.45 75 60 6.82E−05 1.40E−05 20.57 1.83E−061.82E−07 9.93 150 20 1.64E−04 8.39E−06 5.12 5.76E−06 2.45E−07 4.26 15040 1.44E−04 8.34E−06 5.78 4.99E−06 1.72E−07 3.45 150 60 1.17E−041.08E−05 9.23 3.94E−06 1.94E−07 4.91 245 20 1.97E−04 1.12E−05 5.699.30E−06 3.52E−07 3.78 245 40 1.62E−04 1.18E−05 7.30 7.67E−06 1.91E−072.49 245 60 1.35E−04 1.08E−05 7.96 6.11E−06 3.13E−07 5.13 400 202.26E−04 1.40E−05 6.20 1.47E−05 5.26E−07 3.57 400 40 1.94E−04 1.88E−059.69 1.21E−05 3.49E−07 2.88 400 60 1.55E−04 2.24E−05 14.5 8.85E−063.35E−07 3.8

In another variation, instead of applying two distinct pulses, both thecharging and faradaic current of a single electrochemical reaction (i.e.the application of a single pulse) may be used to calculate a correctedglucose value. For example, when a 0.3 V potential is applied to asensor containing ruthenium hexamine, charging current data may becollected at 5 ms I_((T1)) into the reaction along with faradaic currentat 4 s I_((T2)). I_((T1)) and I_((T2)) may be mathematically related toYSI glucose concentrations using a Taylor Series type of least squaresfit. One example of such a Taylor Series fit is depicted in FIG. 4A.

FIG. 4A depicts a three-dimensional Taylor Series fit based on acorrelation of I_((T1)) and I_((T2)) data collected at several glucoseconcentrations and blood hematocrit levels. In FIG. 4A, the variable ofI_((T1)) (depicted along the X axis) and the variable of I_((T2))(depicted along the Y axis) are correlated (with a Taylor Series leastsquares best fit, for example) with the known sample glucoseconcentration levels (the (mg/dl) concentration values along the Z axis)resulting in the surface plot displayed. Thereafter, final glucoseconcentration values (mg/dl) of the samples were obtained by inputtingthe I_((T1)) and I_((T2)) values into the Taylor Series fit.

The resulting glucose values displayed reduced bias with respect tomeasured YSI values (mg/dl). FIG. 4B is a graph depicting the percentbias of the calculated glucose concentration values using the singlepulse technique described in the preceding two paragraphs. As seen inFIG. 4B, the bias of the corrected glucose values are depicted forsamples at multiple concentration values each at various hematocritlevels. Samples having glucose concentration levels of 75 mg/dl, 150mg/dl, 245 mg/dl, and 400 mg/dl were tested, each at three differenthematocrit percentage levels. The resulting % bias of calculatedcorrected glucose values deviating from the YSI values are shown in FIG.4B to be within +15% and −15%.

Table two directly below presents raw data from the above describedone-pulse method charging vs. faradaic current measurement. TABLE 2 OnePulse Data YSI P1 (5 ms) P2 (4 s) Glucose HCT (mg/dl) Ave StDev % CV AveStDev % CV 75 20 80.9 −1.55E−04 8.34E−06 5.38 −3.70E−06 9.15E−08 2.47 7540 73.6 −1.19E−04 1.54E−05 12.93 −2.84E−06 9.85E−08 3.46 75 60 78.9−1.00E−04 8.20E−06 8.18 −2.20E−06 1.90E−07 8.66 150 20 163.3 −2.14E−041.61E−05 7.53 −7.08E−06 2.89E−07 4.09 150 40 166.5 −1.80E−04 1.52E−058.43 −6.01E−06 1.89E−07 3.14 150 60 165.3 −1.36E−04 9.16E−06 6.74−4.41E−06 2.16E−07 4.91 245 20 237.9 −2.47E−04 1.57E−05 6.36 −1.08E−056.70E−07 6.20 245 40 263.3 −2.05E−04 2.15E−05 10.46 −8.85E−06 3.66E−074.13 245 60 260.5 −1.54E−04 1.89E−05 12.30 −6.59E−06 2.76E−07 4.18 40020 408.1 −2.85E−04 1.89E−05 6.64 −1.71E−05 8.01E−07 4.68 400 40 403.2−2.30E−04 2.41E−05 10.51 −1.29E−05 8.00E−07 6.23 400 60 420.0 −1.87E−041.74E−05 9.32 −9.05E−06 4.89E−07 5.40

In another aspect of this system and method, analyte concentrationvalues may be determined directly from the ratios of pulse 1 currentresponse (taken in this instance at t=2 ms) (P1) and pulse 2 currentresponse (taken in this instance at t=4 s) (P2). With reference to FIG.5, a graph is provided depicting the resulting ratio of P1/P2 forsamples at multiple concentration values, and each at various hematocritlevels. Samples having glucose concentration levels of 100 mg/dl, 245mg/dl, 400 mg/dl, and 600 mg/di were tested, and each at three differenthematocrit percentage levels. As seen in FIG. 5, the resulting ratioswere revealed to not be dependent on hematocrit level variations, asevidenced by the relatively constant ratio for each concentration line.Importantly, however, this ratio is revealed to be dependent on theactual glucose concentration of the sample.

FIG. 6, for example, provides a graph of P1/P2 current ratios versus YSIglucose values in mg/dl. This plot indicates that there is a correlationbetween experimentally measured P1/P2 current ratios and the actual YSIglucose concentration sample values. Accordingly, statistical methodscan be used to mathematically convert the measured ratio to a particularglucose concentration value.

Secondary Redox Probe Hematocrit Correction Approach

As noted earlier, during a sample test, the glucose dehydrogenaseinitiates a reaction that oxidizes the glucose to glucono-1,5-lactoneand reduces [Ru(NH₃)₆]³⁺ to [Ru(NH₃)₆]²⁺. When an appropriate voltage isapplied to a working electrode, relative to a counter electrode, the[Ru(NH₃)₆]²⁺ is oxidized to [Ru(NH₃)₆]³⁺, thereby generating a currentthat is related to the glucose concentration in the blood sample. Thecurrent generated is necessarily dependent on the glucose concentrationof the sample. Using this relationship, the glucose level can bedisplayed using a simple correlation algorithm. As also noted above,however, the particular blood hematocrit level can erroneously affect aresulting analyte concentration measurement. Accordingly, an additionalmethod of hematocrit correction has been developed based on the additionof a secondary redox probe (“SRP”) into strip chemistry. For purposes ofthis disclosure, “redox probe” means a substance capable being oxidizedand/or reduced.

In the following disclosure, the measurement technique examined ismulti-step chronoamperometry. However, there are other types ofmeasurement that would be amenable to use in the invention. For examplesquare wave voltammetry, differential pulse amperometry, and cyclicvoltammetry are all contemplated to be viable means of measurement inthe invention. It is not the intention to limit the scope of thisinvention to a particular measurement method.

The particular secondary redox probe can comprise an additional electronmediator substance capable of undergoing an electrochemical redoxreaction. Accordingly, in the same manner as the ruthenium hexaaminemediator mentioned above, the secondary redox probe substance generatesa current in response to the application of a voltage pulse. Thesecondary redox probe, however, differs from the ruthenium hexaamine(i.e. the primary redox probe), or the other mediators cited above, inthat the current generated is instead unrelated to the glucoseconcentration, but still dependent on the particular blood hematocritlevel of the sample.

Accordingly, the electrochemical signal produced by the SRP will be afunction of the hematocrit of the sample, but not glucose dependant, andit will therefore function as an internal standard for hematocritevaluation. This information can be used to correct the glucose signalfor the hematocrit effect as will be described below.

Some of the classes of compounds that could function as a SRP includetransition metal complexes, organometallics, organic dyes and otherorganic redox-active molecules. The following is an exemplary list ofcharacteristics for the SRP. Although preferred, it is not required thatthe SRP exhibit all of the following characteristics.

-   -   The SRP should not interfere with the glucose measurement (i.e.,        limited interaction with the enzyme, mediator, or glucose).    -   The SRP should be oxidized or reduced in a potential range that        can be easily distinguished from that of the mediator.    -   The SRP should be soluble in the strip chemistry formulation.    -   The SRP should not degrade the stability of the sensor, or any        other performance parameter.

For an electrochemically active compound to be useful as an SRP, itshould have a potential distinctly different from the primary mediator,but not so extreme that measuring it would result in a noisy signal dueto interference. For example, when ruthenium hexaamine is used as themediator, there are two preferable (but not required) ‘windows’ in thepotential range. In an oxidation based approach, one of the windows isfrom about 0.3 to approximately 0.9V. The second window is thereduction-based technique, and extends from approximately −0.15V to−0.5V. It is important to remember that the numbers cited here are onlyfor a very specific example, and should not be construed as a generalrule. There may be cases where an SRP that has a peak at 0.2V, or atother magnitudes, would be perfectly acceptable. The actual range of thewindows is dependent on the potential required for the primarymeasurement.

Beyond the scope of hematocrit dependence, potential ranges, and apreference for avoiding interference with the primary measurement, thereare few restrictions on what exactly can be used as an SRP. This enablesthe use of a wide variety of substances, including, but not limited to:simple organics, macromolecules, functionalized microbeads, transitionmetal complexes, nanoparticles, and simple ions.

The SRP is used during a sample measurement by applying a two-steppotential waveform. In the first step, the signal of the primary probeis measured on the working electrode in the standard manner. After thisinitial pulse, a second, different potential pulse, is applied to theworking electrode. This second pulse is designed to measure the signalof the Secondary Redox Probe (“SRP”). The signal is then processed togive a factor that can be compared to a standard value. This will allowthe meter's software to correct the value of the primary measurement.The pulses can be either negative potential (reduction), or positivepotential (oxidation). The preferred type of SRP depends on the primaryprobe used. In the case of the sensors in this specific embodiment, anoxidation-based SRP is advantageous in that an oxidation-based SRP iseasier to implement than the reduction SRP because the primarymeasurement step is the same as the SRP detection step, thus allowingthe SRP measurement to occur on the same set of electrodes as that usedby the primary measurement.

The use of an oxidation based SRP therefore obviates the need to use thefill detect electrodes to form a four-electrode system which would berequired for a reduction-based SRP. This simplifies meter design andprovides other advantages as well. For instance, since the electrodesmeasuring the SRP are the same as those used in the primary measurement,and on the same time scale, in the same sample, it very accuratelyreflects the conditions experienced by the primary redox probe.

Using a reduction based SRP for an oxidation-based system, however, iscertainly possible. Reduction measurements would be conducted on filldetect electrodes by applying a two-step potential waveform. In thisexample, in the first step, ruthenium(III) that is present in the samplewould be reduced to ruthenium(II) so that it does not interfere with themeasurement of the SRP. The second step would be to a more negativepotential at which the SRP is reduced. This signal would then bemeasured to determine hematocrit correction. As noted above, the SRPshould have a reduction potential that is significantly different fromthe reduction potential of the primary mediator (i.e. ruthenium(II) forexample). The SRP potential should be negative enough to completelyreduce the SRP, while not being so negative that it starts to causelarge amounts of background noise. Signal measured with the reductionapproach can become limited by the amount of Ru(II) that is present atthe electrode that serves as the counter electrode, and is glucosedependent at low glucose levels.

In the case of oxidation, the same two-step potential approach could beutilized. In this case, the measurement could be conducted using theprimary measurement anode as the working electrode. The first potentialstep would oxidize ruthenium(II) resulting from the glucose reaction.The potential would then be increased to a higher magnitude required foroxidation of the SRP.

FIG. 7 is a cyclic voltammogram associated with an SRP electron mediatorwhere brilliant cresyl blue, an organic dye, is the selected SRPsubstance. Comparing FIG. 7 and FIG. 1 demonstrates that brilliantcresyl blue has at least a reduction peak significantly different fromthat of the ruthenium hexaamine mediator. Therefore, when a rutheniummediator is used as the primary probe, an SRP of brilliant cresyl bluewill be easily distinguishable from the primary probe in a reductionbased measurement. Therefore, during a measurement, the rutheniumhexaamine mediator can be reduced after the application of a firstpotential pulse and the brilliant cresyl blue mediator can be reducedlater after the application of a second different potential pulse.Brilliant cresyl blue is in this case used as a reduction based SRP.

FIG. 8A is a linear sweep voltammogram associated with another potentialSRP electron mediator, according to an embodiment of the presentdisclosure. FIG. 8A depicts a linear sweep voltammogram of the substancegentisic acid (2,5-dihydroxybenzoic acid). Comparing the gentisic acidpeak (the leftmost peak), to the Ruthenium peak (the right peak)demonstrates that gentisic acid has at least an oxidation peak (e.g. atapproximately 0.81 volts) significantly different from that of theruthenium hexaamine mediator. Therefore, when a ruthenium mediator isused as the primary probe, an SRP of gentisic acid will be easilydistinguishable from the primary probe in an oxidation basedmeasurement. Therefore, during a measurement, the ruthenium hexaaminemediator can be oxidized during the application of a first potentialpulse and the gentisic acid mediator can be oxidized later during theapplication of a second different potential pulse. The foregoingvoltammograms successfully demonstrate the use of simple organiccompounds as SRPs.

Concentration of the SRP used is dependent on the specific SRP inquestion. Many times, the concentration is limited by specificattributes of the SRP or the chemistry. For instance, the SRP may onlybe soluble to a certain concentration, or it may start to affect theprimary measurement at higher concentrations. Also important to note isthe voltage used to measure the SRP. For voltages with a high magnitude,more background will be produced (due to more interferants beingmeasured), and thus a higher concentration of SRP may be needed toeffectively make negligible the background noise contribution.Conversely, an SRP that demonstrates a very intense signal may onlyrequire that a small amount be added to observe an adequate signal.

For the purposes of the SRPs mentioned in this embodiment, 5-20 mM ofSRP mixed into the chemistry formulation seems to be sufficient tocreate an adequate signal without unwanted side effects such asalteration of viscosity and consistency of the chemistry solution, orinterference with the primary measurement.

Since the SRP method relies on a voltage distinctly different from thatof the primary mediator, it may be affected by additional interferantsthat would not necessarily affect the primary measurement. Interferantswill tend to result in an overcorrected hematocrit value (i.e., lowerthan the actual). This is due to the interferant(s) increasing theapparent concentration of the SRP by contributing to the currentmeasured at the SRP detection step. A higher concentration of an SRPwill tend to give a lower response value. This response value iscalculated and not a direct measurement.

For one study, two concentrations of the SRP gentisic acid were used inthe biosensor chemistry, 10 mM and 20 mM. As evidenced in tables 3 and 4below, levels for interferants spiked into the blood were atFDA-mandated levels or above. As can be seen in the tables 3 and 4, the20 mM concentration seems less affected by interferants than the 10 mM.Thus, it may be advantageous to use the highest amount of SRP possiblewithout affecting the primary measurement or going above the saturationpoint of the chemistry involved.

The Salicylate is easily the interferant with the most impact on themeasurement. The other interferants do not register outside the marginof error for the 20 mM. For the 10 mM, the acetaminophen and theascorbic acid register slightly, but their effect is not as pronouncedas that of salicylate. In terms of the effect on the actual correction,the salicylate, being the most noticeable, could generate a measuredshift of approximately 10 hematocrit points for the 10 mM gentisic acidformulation, and 4-6 points for the 20 mM gentisic acid formulation.TABLE 3 20 mM SRP concentration Interferant Response % Bias from ControlControl 0.1394 0.0 Acetaminophen 0.1401 −0.5 Ascorbic Acid 0.1393 0.1Salicylate 0.1347 3.4 Uric Acid 0.1366 2.1

TABLE 4 10 mM SRP concentration Interferant Response % Bias from ControlControl 0.1989 0.0 Acetaminophen 0.1936 2.7 Ascorbic Acid 0.1935 2.7Salicylate 0.1867 6.2 Uric Acid 0.1987 0.1

In the SRP method, the second pulse potential is carefully selected.Biological fluids, such as, for example, blood, are very complexmatrices, and many interferants may be present. Interferants may cause ashift in the SRP signal, which would lead to an erroneous correction. Anerroneous measurement could result in a health risk for the end user.Therefore, it is advantageous to use an SRP substance with as low aredox potential magnitude as possible for a given measurement. Thereasoning for this is that at lower redox potential magnitudes, less ofthe possible pool of interferants undergo redox reactions. Therefore,the resultant response is less likely to be erroneous due to the effectof unintended redox reactions occurring in the interferants.

At the same time, a basic requirement for an SRP is that it have apotential distinctly different from that of the primary redox probe.Therefore, the lower boundary for the magnitude of a particular SRPcandidate's redox potential for any particular system would be thepotential at which the primary probe is measured.

For purposes of exposition, two oxidation-based SRP substances arecompared in FIG. 8B. In FIG. 8B, one SRP is gentisic acid, disclosedpreviously in FIG. 8A. The other is 2,3,4-trihydroxybenzoic acid, aderivative of gentisic acid. These two SRPs are structurally similar,but 2,3,4-trihydroxybenzoic acid has a lower redox potential. The linearsweep voltammograms of FIG. 8B show two blood samples, one containinggentisic acid chemistry (the curve exhibiting a Y-axis value of about−3.4 at a potential of 0.9), and one containing 2,3,4-trihydroxybenzoicacid chemistry (the curve exhibiting a Y-axis value of about −0.7 at apotential of 0.9). Differences in magnitude of the peaks should beignored, as the scan rates is five times as slow for the2,3,4-trihydroxybenzoic acid sweep, resulting in lower magnitude signal.An examination of FIG. 8B reveals that 2,3,4-trihydroxybenzoic acid hasa redox peak near 0.63V, while gentisic acid has a peak near 0.83V. This0.2V difference in potential can be significant. A chronoamperometricexamination of the background signal can reveal the difference in redoxpeaks.

FIG. 8C shows the results of a trial using samples with 3 separatehematocrits at a 245 mg/dL glucose concentration and2,3,4-trihydroxybenzoic acid as the SRP detected at 0.65V. The resultsare comparable to gentisic acid. Therefore, in the absence of evidenceof disadvantageous properties, 2,3,4-trihydroxybenzoic acid would beseen as useful alternative to gentisic acid as the SRP substance since2,3,4-trihydroxybenzoic acid exhibits a lower redox potential magnitude.

As noted above, two approaches can be used for detection of the SRP:reduction and oxidation. In the case of oxidation-based systems,reduction measurements would be conducted on fill detect electrodes byapplying a two-step potential waveform. If the system in question were areduction-based biosensor, a reduction-based SRP would be the moreadvantageous, and would be conducted on the primary electrodes. In thefirst step, the primary mediator, in this case ruthenium(III), that ispresent in the sample would be reduced to ruthenium(II) so that it doesnot interfere with the measurement of the second redox probe. The secondstep would be to a more negative potential at which the second redoxprobe is reduced. This signal would then be measured to determinehematocrit correction. In this case, the SRP should have a reductionpotential that is significantly different from the reduction potentialof ruthenium(III). This potential should be negative enough tocompletely reduce the SRP, while not being so negative that it starts tocause large amounts of background noise.

In the case of oxidation, the same two-step potential approach could beutilized. In this case, the measurement could be conducted using theanode as the working electrode. The first potential step would oxidizeruthenium(II) resulting from the glucose reaction. The potential wouldthen be increased to a higher value required for oxidation of thesecondary redox probe.

FIG. 9 depicts a particular potential input waveform applied at theworking electrode relative to a counter electrode, according to anembodiment of the present disclosure. As seen in FIG. 9, in oneembodiment the pulsing method for SRPs consists of three steps. Thefirst step is optional, and is referred to as mixing time, or wait time.Zero potential is applied to the electrodes, and this essentially givesthe reaction cell contents time to dissolve and mix evenly. This is notrequired for the SRP method, but is used in some embodiments in order toprovide optimal reaction conditions. The next step is the primary redoxprobe measurement step, also called the suppression step.

The suppression step establishes a current baseline for the subsequentSRP step. Since in the example cited, the SRP measurement is performedon the same electrode pair as our primary measurement (i.e. in thisexample both are oxidation based reactions), this step has a dualpurpose. It is both a means of establishing an SRP baseline and it isalso the primary measurement. As seen in FIG. 9, the first step appliesa constant voltage pulse of about 0.30 volts for about 4 seconds. Theprimary measurement step can last any amount of time, but it is found tobe advantageous for it to be at least 3-4 seconds long. Very shortdetection steps result in increased error in both the primary and SRPmeasurements.

The final step is the secondary redox probe measurement step. Thevoltage is changed and the current response generated by the SRP ismeasured. This, along with the baseline, is entered into an equation anda value that describes the hematocrit level is obtained. As seen in FIG.9, in one example, the second step applies a constant voltage pulse ofabout 0.85 volts for a predetermined span of time. The SRP step can bealmost any length of time. However, it is most advantageous to keep thetest time as short as possible for the consumer, thus a test time of 0.1s to 1 s would be considered typical for the purposes of the examplescited above. Again, shorter times are possible, and have been shown tomeasure differences in hematocrit. However, this short measurement mayresult in increased equipment cost.

FIG. 10 is a graph depicting the change in current response over timeduring the application of the input waveform of FIG. 9 in a samplemeasurement using a primary redox probe and a secondary redox probe(“SRP”). The graph of FIG. 10 is a measurement of the current responseof the reaction cell due to the application of a two potential waveform.Therefore, time 0 in FIG. 10 corresponds to time 3.0 in FIG. 9. Thefollowing description provides several exemplary methods of using theSRP data to determine a hematocrit correction factor. Numerous methods,however, can be used to process the data resulting from the inputwaveform of FIG. 9 and the following examples are intended to benon-limiting.

In one embodiment, the current response signal, such as the one depictedin FIG. 10, is measured at a specific point during the time of thesecond pulse. The magnitude of that measurement is subtracted from themagnitude of the current response signal measured at a specific pointduring the first pulse. This process can be described by the followingequation parameters. Two voltage potential pulses are applied to thereaction cell. A first pulse (of a predetermined voltage magnitude) isapplied for the time interval from time zero to time X. A second pulse(of a second predetermined voltage magnitude) is then applied for thetime interval described by the range of time X to time X+Y.

Two current response measurements are recorded at two times, time t₁ andtime t₂ where 0≦t₁≦X and X≦t₂≦X+Y. The two current response values aredescribed as I(t₁) and I(t₂). Therefore, in the numerical processdescribed above, a hematocrit correction factor is obtained bysubtracting I (t₂) from I (t₁), giving a value V. Value V is thencompared with a known standard to determine the particular hematocritcorrection factor.

In an additional method, the magnitude of the current response from thesecond pulse is recorded at two points and the slope between those twopoints is determined. This slope is divided by the magnitude of thecurrent response value measured at the end of the first pulse. Thisvalue obtained is then compared with a known standard to determine theparticular hematocrit correction factor. Accordingly, in this method,three current response values are recorded for mathematical analysis.

This approach is detailed in FIG. 10, where the current response isrecorded at points A, B, and C depicted therein. The current responsevalue of point A is taken right at the end of the first pulse and thecurrent response values for points B and C are recorded during thesecond pulse. This process can be described by the following equationparameters. Just as in the previous process, two voltage potentialpulses are applied to the reaction cell. A first pulse (of apredetermined voltage magnitude) is applied for the time interval fromtime zero to time X. A second pulse (of a second predetermined voltagemagnitude) is then applied for the time interval described by the rangeof time X to time X+Y.

Three current response measurements are recorded at three times, timet₁, time t₂, and time t₃ where 0≦t₁≦X and X≦t₂≦t₃≦X+Y. The two currentresponse values are described as I(t₁), I_((t2)), and I(t₃) (e.g. thecurrent response values for points A, B, and C respectively). As notedabove, the slope between points B and C is calculated and divided by themagnitude of the current response value measured at the end of the firstpulse. This new value, value V can be described by the equation:$\frac{{{{I\left( {t\quad 3} \right)} - {I\left( {t\quad 2} \right)}}}/{{{t\quad 3} - {t\quad 2}}}}{{I\left( {t\quad 1} \right)}}$

-   -   where the numerator defines an absolute value of the slope        between points B and C and the denominator defines the absolute        value magnitude of the current response value measured at the        end of the first pulse.

Therefore, in the numerical process described above, a hematocritcorrection factor is obtained by deriving Value V, according the aboveequation. Since the SRP response is dependent on the blood hematocritlevel, a comparison of SRP signal and the primary signal (as provided ineach of the methods described) yields a value (i.e. value V) that can beused to correct for any errors due to the blood hematocrit level of thesample. The Value V, is then compared with a known standard to determinethe particular hematocrit correction factor for this embodiment.

This measurement may be further refined by taking into account the slopeof the signal resulting from the primary measurement. In this example, 4sample points are taken into consideration for the measurement, T1, T2,T3, and T4. The first is between or equal to 0 and X. The second is alsobetween or equal to 0 and X, but T1<T2. T3 is between or equal to X andY, as is T4. Again, T3<T4. An equation that can be used to describe thisis (T3−(T4−(T1−T2)))/(T3−A*(2*T2−T1)), where A is a constant. Since Timeis constant, it is not included in the aforementioned equation, in orderto simplify computation. This approach can be shown in FIG. 11, wherethe first curve represents the primary analyte measurement (e.g.glucose) and the second curve represents the SRP response signal. FIG.11 is an exemplary current vs. time profile that illustrates the currentreturned from a gentisic acid SRP test. The first (left-hand) decay isthe current generated from the 0.3V pulse, and is the primarymeasurement. The second (right-hand) decay is the SRP pulse at 0.85V,which is suitable for measuring gentisic acid. In this figure, there arefour points marked, which correspond to a particular method formeasuring the SRP response. This system is suitable for gentisic acid.

The above measurements may be altered to take into account bias based onglucose level. In biosensors, it can occur that the bias induced byhematocrit can be more severe depending on the concentration of thetarget analyte. High concentrations of the analyte may have a moresevere bias. To correct for this, a function is needed that increasesthe intensity of the SRP correction effect, but that does not shift themedian point. This can be done by altering the second step in the SRPcorrection process, the comparison of the experimental value to thenominal value. This comparison can be raised to a power that ispartially dependent on the current value generated during the primarymeasurement. This could, for example, take the form(V_(nominal)/V_(experimental))^((B+C*T)), where B and C are numericalconstants and T is the value of the current at some time during theprimary measurement pulse. V_(nominal) is the nominal value of the SRPcorrection factor. V_(experimental) is the experimental value obtainedfor the SRP for a particular sample. The constants can be refined togive good hematocrit correction across a wide range of analyteconcentrations. FIGS. 12 and 13 show one set of data that have beentreated using gentisic acid as the SRP in two separate methods. Theresults in FIG. 12 are based on a straight linear correction based onthe four-point method outlined in the above paragraphs. The results inFIG. 13 were obtained by adding an exponential correction function tothe comparison equation. As can be seen, exponential correction is muchmore effective in correcting high concentrations, while not sacrificingaccuracy at low concentrations. Therefore, it is most preferable toinclude this in the SRP calculations.

FIG. 14 is a graph depicting the dependence of an SRP mediator,brilliant cresyl blue, on the particular hematocrit level of blood. Thegraph depicts the current response values for samples with aconcentration level of 400 mg/dl. The samples were measured athematocrit concentration levels of 0, 42, and 58. As seen in FIG. 14,there is a linear relationship between the measured current response andthe hematocrit level of the sample. Accordingly, the measured SRPresponse is clearly dependent on the hematocrit sample level.

FIG. 15 depicts the relationship between the measured analyte signalmagnitude and the actual sample analyte concentration using multipleconcentrations of the SRP in the test strip chemistry. In the experimentdepicted, strips containing cresyl blue SRP concentrations of 0 mM, 5mM, and 10 mM added to the standard chemistry formulation were each handdispensed and tested with samples having 0, 75, and 600 mg/dL glucoseconcentrations. The results illustrate that the addition of the SRP doesnot erroneously interfere with the glucose measurement.

FIG. 16 is a graph showing the derived relationship between a calculatedSRP factor and the hematocrit of the sample. In this case, a higher SRPvalue is indicative of lower hematocrit.

With reference to the drawings, FIGS. 17 and 18 show a test strip 10, inaccordance with an exemplary embodiment of the present invention. Teststrip 10 preferably takes the form of a generally flat strip thatextends from a proximal end 12 to a distal end 14. Preferably, teststrip 10 is sized for easy handling. For example, test strip 10 canmeasure approximately 35 mm long (i.e., from proximal end 12 to distalend 14) and approximately 9 mm wide. However, the strip can be anyconvenient length and width. For example, a meter with automated teststrip handling may utilize a test strip smaller than 9 mm wide.Additionally, proximal end 12 can be narrower than distal end 14 inorder to provide facile visual recognition of the distal end. Thus, teststrip 10 can include a tapered section 16, in which the full width oftest strip 10 tapers down to proximal end 12, making proximal end 12narrower than distal end 14. As described in more detail below, the userapplies the blood sample to an opening in proximal end 12 of test strip10. Thus, providing tapered section 16 in test strip 10, and makingproximal end 12 narrower than distal end 14, assists the user inlocating the opening where the blood sample is to be applied. Further,other visual means, such as indicia, notches, contours or the like arepossible.

As shown in FIG. 18, test strip 10 can have a generally layeredconstruction. Working upwardly from the bottom layer, test strip 10 caninclude a base layer 18 extending along the entire length of test strip10. Base layer 18 can be formed from an electrically insulating materialand has a thickness sufficient to provide structural support to teststrip 10. For example, base layer 18 can be a polyester material about0.35 mm thick.

According to the illustrative embodiment, a conductive layer 20 isdisposed on base layer 18. Conductive layer 20 includes a plurality ofelectrodes disposed on base layer 18 near proximal end 12, a pluralityof electrical contacts disposed on base layer 18 near distal end 14, anda plurality of conductive regions electrically connecting the electrodesto the electrical contacts. In the illustrative embodiment depicted inFIGS. 17-18, the plurality of electrodes includes a working electrode22, a counter electrode 24, a fill-detect anode 28, and a fill-detectcathode 30. Correspondingly, the electrical contacts can include aworking electrode contact 32, a counter electrode contact 34, afill-detect anode contact 36, and a fill-detect cathode contact 38. Theconductive regions can include a working electrode conductive region 40,electrically connecting working electrode 22 to working electrodecontact 32, a counter electrode conductive region 42, electricallyconnecting counter electrode 24 to counter electrode contact 34, afill-detect anode conductive region 44 electrically connectingfill-detect anode 28 to fill-detect contact 36, and a fill-detectcathode conductive region 46 electrically connecting fill-detect cathode30 to fill-detect cathode contact 38. Further, the illustrativeembodiment is depicted with conductive layer 20 including an auto-onconductor 48 disposed on base layer 18 near distal end 14.

The next layer in the illustrative test strip 10 is a dielectric spacerlayer 64 disposed on conductive layer 20. Dielectric spacer layer 64 iscomposed of an electrically insulating material, such as polyester.Dielectric spacer layer 64 can be about 0.100 mm thick and coverportions of working electrode 22, counter electrode 24, fill-detectanode 28, fill-detect cathode 30, and conductive regions 40-46, but inthe illustrative embodiment does not cover electrical contacts 32-38 orauto-on conductor 48. For example, dielectric spacer layer 64 can coversubstantially all of conductive layer 20 thereon, from a line justproximal of contacts 32 and 34 all the way to proximal end 12, exceptfor a slot 52 extending from proximal end 12. In this way, slot 52 candefine an exposed portion 54 of working electrode 22, an exposed portion56 of counter electrode 24, an exposed portion 60 of fill-detect anode28, and an exposed portion 62 of fill-detect cathode 30.

A cover 72, having a proximal end 74 and a distal end 76, can beattached to dielectric spacer layer 64 via an adhesive layer 78. Cover72 can be composed of an electrically insulating material, such aspolyester, and can have a thickness of about 0.1 mm. Additionally, thecover 72 can be transparent.

Adhesive layer 78 can include a polyacrylic or other adhesive and have athickness of about 0.013 mm. Adhesive layer 78 can consist of sectionsdisposed on spacer 64 on opposite sides of slot 52. A break 84 inadhesive layer 78 extends from distal end 70 of slot 52 to an opening86. Cover 72 can be disposed on adhesive layer 78 such that its proximalend 74 is aligned with proximal end 12 and its distal end 76 is alignedwith opening 86. In this way, cover 72 covers slot 52 and break 84.

Slot 52, together with base layer 18 and cover 72, defines a samplechamber 88 in test strip 10 for receiving a blood sample for measurementin the illustrative embodiment. Proximal end 12 of slot 52 defines afirst opening in sample chamber 88, through which the blood sample isintroduced into sample chamber 88. At distal end 70 of slot 52, break 84defines a second opening in sample chamber 88, for venting samplechamber 88 as sample enters sample chamber 88. Slot 52 is dimensionedsuch that a blood sample applied to its proximal end 68 is drawn intoand held in sample chamber 88 by capillary action, with break 84 ventingsample chamber 88 through opening 86, as the blood sample enters.Moreover, slot 52 can advantageously be dimensioned so that the bloodsample that enters sample chamber 88 by capillary action is about 1micro-liter or less. For example, slot 52 can have a length (i.e., fromproximal end 12 to distal end 70) of about 0.140 inches, a width ofabout 0.060 inches, and a height (which can be substantially defined bythe thickness of dielectric spacer layer 64) of about 0.005 inches.Other dimensions could be used, however.

A reagent layer 90 is disposed in sample chamber 88. Preferably, reagentlayer spreads uniformly throughout the sample cavity. Reagent layer 90includes chemical constituents to enable the level of glucose in theblood sample to be determined electrochemically. Thus, reagent layer 90may include an enzyme specific for glucose and a mediator, as describedabove. In addition, reagent layer 90 may also include other components,such as the secondary redox probe (SRP) materials, buffering materials(e.g., potassium phosphate), polymeric binders (e.g.,hydroxypropyl-methyl-cellulose, sodium alginate, microcrystallinecellulose, polyethylene oxide, hydroxyethylcellulose, and/or polyvinylalcohol), and surfactants (e.g., Triton X-100 or Surfynol 485).

With these chemical constituents, reagent layer 90, including thesecondary redox probe material, reacts with glucose in the blood samplein the manner described throughout this application.

As depicted in FIG. 18, the arrangement of the various layers inillustrative test strip 10 can result in test strip 10 having differentthicknesses in different sections. In particular, among the layers abovebase layer 18, much of the thickness of test strip 10 can come from thethickness of spacer 64. Thus, the edge of spacer 64 that is closest todistal end 14 can define a shoulder 92 in test strip 10. Shoulder 92 candefine a thin section 94 of test strip 10, extending between shoulder 92and distal end 14, and a thick section 96, extending between shoulder 92and proximal end 12. The elements of test strip 10 used to electricallyconnect it to the meter, namely, electrical contacts 32-38 and auto-onconductor 48, can all be located in thin section 94. Accordingly, theconnector in the meter can be sized and configured to receive thinsection 94 but not thick section 96, as described in more detail below.This can beneficially cue the user to insert the correct end, i.e.,distal end 14 in thin section 94, and can prevent the user frominserting the wrong end, i.e., proximal end 12 in thick section 96, intothe meter.

Although FIGS. 17 and 18 illustrate an illustrative embodiment of teststrip 10, other configurations, chemical compositions and electrodearrangements could be used.

Different arrangements of fill-detect electrodes 28 and 30 can also beused. In the configuration shown in FIGS. 17 and 18, fill-detectelectrodes 28 and 30 are in a side-by-side arrangement. Alternatively,fill-detect electrodes 28 and 30 can be in a sequential arrangement,whereby, as the sample flows through sample chamber 88 toward distal end70, the sample contacts one of the fill-detect electrodes first (eitherthe anode or the cathode) and then contacts the other fill-detectelectrode.

As depicted in the Figures, fill-detect electrodes 28 and 30 areadvantageously located on the distal side of reagent layer 90. In thisarrangement, the sample introduced into the sample chamber 88 will havetraversed reagent layer 90 before reaching fill-detect electrodes 28 and30. This arrangement beneficially allows the fill-detect electrodes 28and 30 to indicate not only whether sufficient blood sample is presentin sample chamber 88, but also when, concomitantly, the blood sample hassufficiently mixed with the chemical constituents of reagent layer 90.Other configurations are of course possible.

Test Strip Array Configuration

Test strips can be manufactured by forming a plurality of strips in anarray along a reel or web of substrate material. The term “reel” or“web” as used herein applies to continuous webs of indeterminate length,or to sheets of determinate length. The individual strips, after beingformed, can be separated during later stages of manufacturing. Anillustrative embodiment of a batch process of this type is describedinfra. First, an illustrative test strip array configuration isdescribed.

FIG. 19 shows a series of traces 80 formed in a substrate materialcoated with a conductive layer. Traces 80, formed in the exemplaryembodiment by laser ablation, partially form the conductive layers oftwo rows of ten test strips as shown. In the exemplary embodimentdepicted, proximal ends 12 of the two rows of test strips are injuxtaposition in the center of a reel 100. The distal ends 14 of thetest strips are arranged at the periphery of reel 100. It is alsocontemplated that the proximal ends 12 and distal ends 14 of the teststrips can be arranged in the center of reel 100. Alternatively, the twodistal ends 14 of the test strips can be arranged in the center of reel100. The lateral spacing of the test strips is designed to allow asingle cut to separate two adjacent test strips. The separation of thetest strip from reel 100 can electrically isolate one or more conductivecomponents of the separated test strip 10.

As depicted in FIG. 19, trace 80 for an individual test strip forms aplurality of conductive components; e.g., electrodes, conduction regionsand electrode contacts. Trace 80 is comprised of individual cuts made bya laser following a specific trajectory, or vector. A vector can belinear or curvilinear, and define spaces between conductive componentsthat are electrically isolating. Generally a vector is a continuous cutmade by the laser beam.

The conductive components can be partially or entirely defined byablated regions, or laser vectors, formed in the conductive layer. Thevectors may only partially electrically isolate the conductivecomponent, as the component can remain electrically connected to othercomponents following laser ablation. The electrical isolation of theconductive components can be achieved following “singulation,” whenindividual test strips are separated from reel or web 100.

FIG. 19 shows a plurality of electrically isolated working electrodes22. According to the illustrated embodiment, working electrode 22 of anindividual test strip can be electrically isolated from the otherconductive components during the laser ablation process. It is alsocontemplated that other conductive components may be electricallyisolated during the laser ablation process. For example, fill detectelectrodes may be isolated with the addition of one or more vectors.

FIG. 19 also includes registration points 102 at the distal end 14 ofeach test strip on reel 100. Registration points 102 assist thealignment of the layers during the lamination, punching and othermanufacturing processes. It is further contemplated that registrationpoints 102 may be located at locations other than the distal end 14 ofeach test strip trace 80 on reel 100. High quality manufacturing mayrequire additional registration points 102 to ensure adequate alignmentof laminate layers and/or other manufacturing processes, such as, forexample, laser ablation of conductive components, reagent deposition,singulation, etc.

FIG. 20 shows a number of strips forming a card 104 separated from reel100. Card 104 can contain a plurality of test strips 10 or traces 80,and a plurality of conductive components. In the preferred embodimentcard 104 can contain between 6 and 12 test strips 10 or traces 80. Inother embodiments, card 104 can contain a plurality of test strips 10 ortraces 80. In the illustrated embodiment, card 104 can include a lateralarray of test strips 10 or traces 80. In other embodiments, card 104 caninclude an array or arrays of test strips 10 or traces 80 inlongitudinal and/or lateral configurations. It is further contemplatedthat test strips 10 or traces 80 may be in any arrangement on reel 100suitable for manufacturing.

Card 104 contains a plurality of conductive components. Some conductivecomponents can be electrically isolated when the card is removed fromthe reel. As shown in FIG. 20, working electrode 22 is electricallyisolated. Other embodiments could include additional electricallyisolated conductive components not shown in FIG. 21. It may be possibleto analyze properties of the electrically isolated conductive componentsto assess the quality of the manufacturing process and strip chemistryapplication. The efficiency of the quality assessment process can beincreased by testing at least one of the plurality of electricallyisolated conductive components in order to determine a particularcalibration code based on the particular strip chemistry, for example.

Batch Manufacturing of Test Strips

FIGS. 21 through 24 illustrate an exemplary method of manufacturing teststrips. Although these figures shows steps for manufacturing test strip10, as shown in FIGS. 17 and 18, it is to be understood that similarsteps can be used to manufacture test strips having otherconfigurations.

With reference to FIG. 20, a plurality of test strips 10 can be producedby forming a structure 120 that includes a plurality of test striptraces 122 on reel 100. Test strip traces 122 include a plurality oftraces 80, and can be arranged in an array that includes a plurality ofrows. Each row 124 can include a plurality of test strip traces 122.

The separation process can also be used to electrically isolateconductive components of test strip 10. Laser ablation of the conductivelayer may not electrically isolate certain conductive components. Thenon-isolated conductive components may be isolated by the separationprocess whereby test strips are separated from reel 100. The separationprocess may sever the electrical connection, isolating the conductivecomponent. Separating test strip 10 can electrically isolate thecounting electrode 24, fill detect-anode 28 and fill-detect cathode 30.The separation process can complete the electrical isolation ofconductive components by selectively separating conductive components.

Further, the separation process can provide some or all of the shape ofthe perimeter of the test strips 10. For example, the tapered shape oftapered sections 16 of the test strips 10 can be formed during thispunching process. Next, a slitting process can be used to separate thetest strip structures 122 in each row 124 into individual test strips10. The separation process may include stamping, slitting, scoring andbreaking, or any suitable method to separate test strip 10 and/or card104 from reel 100.

FIGS. 21 and 22 show only one test strip structure (either partially orcompletely fabricated), in order to illustrate various steps in apreferred method for forming the test strip structures 122. In thisexemplary approach, the test strip structures 122 in integratedstructure 120 are all formed on a sheet of material that serves as baselayer 18 in the finished test strips 10. The other components in thefinished test strips 10 are then built up layer-by-layer on top of baselayer 18 to form the test strip structures 122. In each of FIGS. 21 and22, the outer shape of the test strip 10 that would be formed in theoverall manufacturing process is shown as a dotted line.

The exemplary manufacturing process employs base layer 18 covered byconductive layer 20. Conductive layer 20 and base layer 18 can be in theform of a reel, ribbon, continuous web, sheet, or other similarstructure. Conductive layer 20 can include any suitable conductive orsemi-conductor material, such as gold, silver, palladium, carbon, tinoxide and others known in the art. Conductive layer 20 can be formed bysputtering, vapor deposition, screen printing or any suitablemanufacturing method. The conductive material can be any suitablethickness and can be bonded to base layer 18 by any suitable means.

As shown in FIG. 21, conductive layer 20 can include working electrode22, counter electrode 24, fill-detect anode 28, and fill-detect cathode30. Trace 80 can be formed by laser ablation where laser ablation caninclude any device suitable for removal of the conductive layer inappropriate time and with appropriate precision and accuracy. Varioustypes of lasers can be used for sensor fabrication, such as, forexample, solid-state lasers (e.g. Nd:YAG and titanium sapphire), coppervapor lasers, diode lasers, carbon dioxide lasers and excimer lasers.Such lasers may be capable of generating a variety of wavelengths in theultraviolet, visible and infrared regions. For example, excimer laserprovides wavelength of 248 nm, a fundamental Nd:YAG laser gives 1064 nm,a frequency tripled Nd:YAG wavelength is at 355 nm and a Ti:sapphirelaser is at approximately 800 nm. The power output of these lasers mayvary and is usually in range 10-100 watts.

The laser ablation process can include a laser system. The laser systemcan include a laser source. The laser system can further include meansto define trace 80, such as, for example, a focused beam, projected maskor other suitable technique. The use of a focused laser beam can includea device capable of rapid and accurate controlled movement to move thefocused laser beam relative to conductive layer 20. The use of a maskcan involve a laser beam passing through the mask to selectively ablatespecific regions of conductive layer 20. A single mask can define teststrip trace 80, or multiple masks may be required to form test striptrace 80. To form trace 80, the laser system can move relative toconductive layer 20. Specifically, the laser system, conductive layer20, or both the laser system and conductive layer 20 may move to allowformation trace 80 by laser ablation. Exemplary devices available forsuch ablation techniques include Microline Laser system available fromLPKF Laser Electronic GmbH (Garbsen, Germany) and laser micro machiningsystems from Exitech, Ltd (Oxford, United Kingdom).

In the next step, dielectric spacer layer 64 can be applied toconductive layer 20, as illustrated in FIG. 22. Spacer 64 can be appliedto conductive layer 20 in a number of different ways. In an exemplaryapproach, spacer 64 is provided as a sheet or web large enough andappropriately shaped to cover multiple test strip traces 80. In thisapproach, the underside of spacer 64 can be coated with an adhesive tofacilitate attachment to conductive layer 20. Portions of the uppersurface of spacer 64 can also be coated with an adhesive in order toprovide adhesive layer 78 in each of the test strips 10. Various slotscan be cut, formed or punched out of spacer 64 to shape it before,during or after the application of spacer layer 64 to conductive layer20. For example, as shown in FIG. 22, spacer 64 can have a pre-formedslot 136 for each test strip structure. In addition, spacer 64 caninclude adhesive sections 66, with break 84 there between, for each teststrip trace 80. Spacer 64 is then positioned over conductive layer 20,as shown in FIG. 23, and laminated to conductive layer 20. When spacer64 is appropriately positioned on conductive layer 20, exposed electrodeportions 54-62 are accessible through slot 136. Thus, slot 52 in teststrip 10 corresponds to that part of slot 136 that remains in test strip10 after the test strip structures are separated into test strips.Similarly, spacer 64 leaves contacts 32-38 and auto-on conductor 48exposed after lamination.

Alternatively, spacer 64 could be applied in other ways. For example,spacer 64 can be injection molded onto base layer 18 and dielectric 50.Spacer 64 could also be built up on dielectric layer 50 byscreen-printing successive layers of a dielectric material to anappropriate thickness, e.g., about 0.005 inches. A preferred dielectricmaterial comprises a mixture of silicone and acrylic compounds, such asthe “Membrane Switch Composition 5018” available from E.I. DuPont deNemours & Co., Wilmington, Del. Other materials could be used, however.

Reagent layer 90 may then be applied to each test strip structure. In apreferred approach, reagent layer 90 is applied by micropipetting anaqueous composition into sample cavity and drying it to form reagentlayer 90. One aqueous composition includes an enzyme specific forglucose, a mediator, and the secondary redox probe (SRP) material.Alternatively, other methods, such as screen-printing, may be used toapply the composition used to form reagent layer 90.

A transparent cover 72 can then be attached to adhesive layer 78. Cover72 may be large enough to cover multiple test strip structures 122.Attaching cover 72 can complete the formation of the plurality of teststrip structures 122. The plurality of test strip structures 122 canthen be separated from each other to form a plurality of test strips 10,as described above.

Quality Control Testing of Test Strips

FIG. 23 shows a further illustrative embodiment of a test stripmanufacturing method. The manufacturing method utilizes a web 200containing conductive layer 20 and base layer 18. Conductive layer 20and base layer 18 can be any suitable material. Web 200 can be anydimension suitable for production of the test strips. Web 200 is passedthrough any suitable device and ablated by process 300.

Ablation 300 can include any suitable ablation process capable offorming conductive components in conductive layer 20. In theillustrative embodiment, ablation 300 is achieved by laser ablation. Theablation process may not electrically isolate all conductive components.For example, counter electrode 24 may not be isolated by laser ablationbut can be isolated by subsequent separation from web 200. In theillustrative embodiment, working electrode 22 is electrically isolatedduring ablation process 300. The counter electrode 24, fill-detect anode28 and fill-detect cathode 30 may not be electrically isolated duringablation process 300. Specifically, subsequent separation process canelectrically isolate the counter electrode 24, fill-detect anode 28 andfill-detect cathode 30.

Web 200 can be passed through any suitable ablation device at speedssufficient to produce an appropriate rate of test strip production. Theablation process can be sufficiently rapid to allow the continuousmovement of web 200 through the laser ablation device. Alternatively,web 200 can be passed through the ablation device in a non-continuous(i.e., start-and-stop) manner.

The properties of the conductive components formed by ablation process300 can be analyzed during or following ablation process 300. Analysisof ablation process 300 can include optical, chemical, electrical or anyother suitable analysis means. The analysis can monitor the entireablation process, or part of the ablation process. For example, theanalysis can include monitoring vector formation to ensure thedimensions of the formed vector are within predetermined toleranceranges.

Quality control analysis, which can be performed during or uponcompletion of the manufacturing process, can also include monitoring theeffectiveness and/or efficiency of the vector formation process. Inparticular, the width of the resulting vectors can be monitored toensure acceptable accuracy and precision of the cuts in conductive layer20. For example, the quality of the laser ablation process can beanalyzed by monitoring the surface of conductive layer 20 and/or baselayer 18 following ablation. Partial ablation of base layer 18 canindicate that the laser power is set too high or the beam is travelingtoo slowly. By contrast, a partially ablated conductive layer mayindicate insufficient laser power or that the beam is traveling tooquickly. Incomplete ablation of gaps may result in the formation ofvectors that are not electrically isolating between conductivecomponents.

In the illustrative embodiment, the dimensions of working electrode 22can be analyzed to determine the quality of the manufacturing process.For example optical analysis (not shown) can monitor the width ofworking electrode 22 to ensure sufficient accuracy of ablation process300. Further, the alignment of working electrode 22 relative toregistration points 102 can be monitored. Optical analysis can beperformed by using VisionPro system from Cognex Vision Systems (Natick,Mass.).

As described above, the ablation process produces an array of teststrips 202 on web 200. Following formation of test strip array 202 andcorresponding conductive components, dielectric spacer 64 is laminatedto conductive layer 20. The spacer lamination process 302 can includeregistration points 102 to correctly align spacer layer 64 withconductive layer 20. Spacer 64 may contain registration points 102corresponding to registration points 102 of test strip array 202. Thecorrect alignment of the layers will position slot 136 over theelectrodes as indicated in FIG. 22, forming a three-layer laminate 204.

Following the formation of three-layer laminate 204, the chemistry canbe applied to three-layer laminate 204 by a chemistry applicationprocess 310. The resulting laminate 208 can contain any appropriatereagent suitable for the specific test strip. The reagent applicationprocess 310 can include any appropriate process, such as, for example,the application of an SRP component.

Following reagent application 310, cover 72 can be applied to laminate208 using any appropriate cover application process 312. Cover 72 may becentered on laminate 208. The resulting laminate 210 can be tested toensure the quality of the cover application process 312. For example,optical means can be used to monitor the alignment of the cover tolaminate 208. Alternatively, laminate 210 can be tested to ensure thequality of any upstream manufacturing process as described previously.Following cover application 312, laminate 210 can be subject to qualitycontrol testing as mentioned above. For example, quality controlanalysis can monitor the effectiveness of the chemistry application.Specifically, optical analysis may be required to determine the extentof reagent covering working electrode 22 and/or counter electrode 24.Alternatively, any previous or upstream manufacturing process can betested following formation of laminate 210. In addition, following theformation of laminate 210 an entire strip lot can be analyzed todetermine a particular lot code to be associated with that particularstrip lot. For example, during process 314, the resulting laminate 210(or even a portion, such as card 104, depicted in FIG. 20) could beanalyzed to determine a lot code that includes information regarding aparticular calibration code used by a meter to produce accurate samplemeasurements. The coded information may be any suitable identifiercontaining batch, lot, manufacturing, and/or other information pertinentto the manufacturing process, test strip 10, and/or the underlyingmeter.

The resulting coded assembled web containing test strips 10 with codednumbers, for example, can be passed into a device to form singulatedtest strips. The singulation process, for example, can includesingulation of the individual test strips and/or any appropriatehandling or packaging process. The singulated test strips (not shown)can be further processed if required. For example, test strips 10 of thecoded assembled web can be singulated and placed in storage vials.

CONCLUSION

In summary, the SRP correction approach has a number of advantages. Itcan be applied to many biosensors, not just oxidation-based glucosesensors. It has a high degree of accuracy and precision in regards tomeasuring and correcting for hematocrit, and can even improve % CVswithin a sample set, enhancing not only hematocrit bias correction, butthe actual overall precision of the measurement device. The amount oftest time added due to the SRP is negligible and is often not noticeableto the end user.

Further, this method negates the need for a complicated table ormultivariate matrix of primary analyte signal versus SRP signal, as evena simple correction algorithm will produce an output which does not varywith respect to analyte signal across a wide range of analyteconcentration, but which does vary consistently and accurately withhematocrit.

While various substances are described as possible candidates for use asan SRP, they are not intended to be limiting of the claimed invention.Unless expressly noted, the particular substances are listed merely asexamples and are not intended to be limiting of the invention asclaimed. Other embodiments of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. It is intended that the specificationand examples be considered as exemplary only, with a true scope andspirit of the invention being indicated by the following claims.

1. A biosensor for measuring a constituent concentration in blood, saidbiosensor comprising: a sample reception region for receiving a bloodsample; and a reaction reagent system comprising: an oxidation-reductionenzyme specific for the constituent; a first electron mediator capableof being reversibly reduced and oxidized such that a firstelectrochemical signal resulting from the reduction or oxidation isrelated to the constituent concentration in the blood sample; a secondelectron mediator capable of undergoing an electrochemical redoxreaction where a second electrochemical signal produced by oxidation orreduction of the second mediator is not directly related to theconstituent concentration in the blood sample; and wherein the secondelectrochemical signal changes based on the hematocrit level of theblood sample.
 2. The biosensor of claim 1, wherein the constituent isglucose.
 3. The biosensor of claim 1, wherein the first mediator is aruthenium containing material.
 4. The biosensor of claim 3, wherein theruthenium containing material comprises hexaamine ruthenium (III)trichloride.
 5. The biosensor of claim 1, wherein the second mediatorcomprises brilliant cresyl blue.
 6. The biosensor of claim 1, whereinthe second mediator comprises gentisic acid (2,5-dihydroxybenzoic acid).7. The biosensor of claim 1, wherein the second mediator comprises2,3,4-trihydroxybenzoic acid.
 8. The biosensor of claim 1, wherein thesecond mediator does not interfere with the first electrochemicalsignal.
 9. The biosensor of claim 1, wherein the second mediator isoxidized or reduced in a potential range distinguishable from that ofthe first mediator.
 10. The biosensor of claim 1, wherein the secondelectron mediator is oxidized or reduced at a potential having amagnitude at least 0.2 volts greater or less than that used to oxidizeor reduce the first electron mediator.
 11. The biosensor of claim 1,wherein the first and second electrochemical signals are electriccurrent signals obtained through multi-step chronoamperometry.
 12. Thebiosensor of claim 1, wherein the first and second electrochemicalsignals are electric current signals obtained through square wavevoltammetry.
 13. The biosensor of claim 1, wherein the first and secondelectrochemical signals are electric current signals obtained throughdifferential pulse amperometry.
 14. The biosensor of claim 1, whereinthe first and second electrochemical signals are electric currentsignals obtained through cyclic voltammetry.
 15. A method fordetermining a constituent concentration in blood, the method comprising:(a) introducing the blood sample into an electrochemical cellcomprising: (i) spaced apart working and counter electrodes; and (ii) aredox reagent system comprising an enzyme; a first electron mediatorcapable of being reversibly reduced and oxidized such that a firstelectrochemical signal resulting from the reduction or oxidation isrelated to the constituent concentration in the blood sample; and asecond electron mediator capable of capable of undergoing anelectrochemical redox reaction where a second electrochemical signalproduced by oxidation or reduction of the second mediator is notdirectly related to the constituent concentration in the blood sampleand changes based on the hematocrit level of the blood sample; (b)obtaining the first electrochemical signal; (c) obtaining the secondelectrochemical signal; (d) determining an initial value correspondingto the constituent concentration of the sample using data from the firstelectrochemical signal; and (e) correcting the initial valuecorresponding to the constituent concentration of the sample to removean effect of the hematocrit level of the sample using a statisticalcorrelation algorithm and data from the second electrochemical signal.16. The method of claim 15, wherein the constituent is glucose.
 17. Themethod of claim 15, wherein correcting the initial value comprises:deriving a preliminary constituent concentration from the first andsecond signals; and multiplying the preliminary constituentconcentration by a correction factor based on the second electrochemicalsignal to derive the constituent concentration in the sample, correctedto offset an effect of the hematocrit level of the blood sample.
 18. Themethod of claim 15, wherein the statistical correlation comprisesdetermining a slope of the second electrochemical signal.
 19. The methodof claim 15, wherein the statistical correlation comprises determining aslope of both the first and second electrochemical signals.
 20. Themethod of claim 15, wherein the first electrochemical signal is obtainedby applying to the electrochemical cell, a first electric potential of amagnitude capable of oxidizing or reducing the first electron mediatorand not capable of oxidizing or reducing the second electron mediator.21. The method of claim 20, wherein the second electrochemical signal isobtained by applying to the electrochemical cell, a second electricpotential of a magnitude capable of oxidizing or reducing the secondelectron mediator and not capable of oxidizing or reducing the firstelectron mediator.
 22. The method of claim 15, wherein the secondelectron mediator is oxidized or reduced at a potential having amagnitude at least 0.2 volts greater or less than that used to oxidizeor reduce the first electron mediator.
 23. The method of claim 15,wherein obtaining the first and second electrochemical signals comprisesusing multi-step chronoamperometry.
 24. The method of claim 15, whereinobtaining the first and second electrochemical signals comprises usingsquare wave voltammetry.
 25. The method of claim 15, wherein obtainingthe first and second electrochemical signals comprises usingdifferential pulse amperometry.
 26. The method of claim 15, whereinobtaining the first and second electrochemical signals comprises usingcyclic voltammetry.
 27. The method of claim 15, wherein the secondelectron mediator comprises brilliant cresyl blue.
 28. The method ofclaim 15, wherein the second electron mediator comprises gentisic acid(2,5-dihydroxybenzoic acid).
 29. The method of claim 15, wherein thesecond electron mediator comprises 2,3,4-trihydroxybenzoic acid.
 30. Amethod for determining the hematocrit corrected concentration of ananalyte in a physiological sample, said method comprising: (a)introducing the physiological sample into an electrochemical cellcomprising: (i) spaced apart working and counter electrodes; and (ii) aredox reagent system comprising an enzyme and a mediator; (b) applying afirst electric potential to the reaction cell and measuring cell currentduring a first 50 milliseconds of the first electric potential as afunction of time to obtain a first time-current transient; (c) applyinga second electric potential to said cell, and measuring cell current asa function of time to obtain a second time-current transient; (d)deriving a preliminary analyte concentration from said first and secondtime-current transients; and (e) multiplying the preliminary analyteconcentration by a hematocrit correction factor based on the first andsecond time-current transient to derive the hematocrit corrected analyteconcentration in said sample; whereby the hematocrit correctedconcentration of said analyte in said sample is determined.
 31. Themethod of claim 30, wherein the first electric potential is a negativeelectric pulse and the second electrical potential is a positiveelectrical pulse.
 32. The method of claim 30, wherein the first electricpotential is an applied pulse having a duration of about 1-10milliseconds.
 33. The method of claim 30, wherein the preliminaryanalyte concentration is determined in part based on a current timetransient value as sampled at an end of the applied pulse of the firstelectric potential.
 34. The method of claim 30, wherein the secondelectric potential is an applied pulse or about 1-4 seconds.
 35. Themethod of claim 30, wherein the preliminary analyte concentration isdetermined in part based on a current time transient value as sampled atan end of the applied pulse of the second electric potential.
 36. Amethod of manufacturing a plurality of test strips, comprising: forminga web containing a conductive layer and a base layer; partially formingsaid plurality of test strips by electrically isolating a first group ofconductive components in the conductive layer using a first process;subsequently forming said plurality of test strips by electricallyisolating a second group of conductive components in the conductivelayer using a second process wherein first and second processes are notthe same; and forming a reagent layer including: an enzyme; a firstelectron mediator capable of being reversibly reduced and oxidized suchthat a first electrochemical signal resulting from the reduction oroxidation is related to the constituent concentration in the bloodsample; and a second electron mediator capable of undergoing anelectrochemical redox reaction where a second electrochemical signalproduced by oxidation or reduction of the second mediator is notdirectly related to the constituent concentration in the blood sampleand changes based on the hematocrit level of the blood sample.
 37. Themethod of claim 36, wherein the web includes a plurality of registrationpoints.
 38. The method of claim 36, wherein the first process includes alaser ablation process.
 39. The method of claim 36, wherein the secondprocess includes a separation process.
 40. The method of claim 39,wherein the separation process includes stamping.
 41. The method ofclaim 39, wherein the separation process includes separating a pluralityof test strips from the web.
 42. The method of claim 37, wherein theplurality of registration points are separated by approximately 9 mm.43. The method of claim 37, wherein the plurality of registration pointsare separated by less than approximately 9 mm.
 44. The method of claim36, wherein the first group of conductive components are separated byless than approximately 9 mm.